The Drosophila lark gene encodes an RRM-type RNA-binding protein that is conserved among flies, mice and humans. Our previous studies of lark indicate that it acts as a repressor protein in the circadian clock signaling (output) pathway which mediates the regulation of adult eclosion (the emergence of adults from pupal cases). Work from the lab also indicates that the clock regulation of eclosion occurs via a rhythm in lark protein abundance. We have recently shown, for example, that lark protein abundance changes during the daily cycle, with steady-state amounts being lowest several hours prior to adult eclosion. Based on our studies, we hypothesize that decreases in levels of the lark repressor lead to activation of the eclosion output pathway and the initiation of the eclosion process. This application proposes studies that will lead to a more complete understanding of lark's role in the clock regulation of adult eclosion. We propose to: (1) Further characterize the rhythm in lark protein abundance and determine whether it depends on the activity of clock proteins such as Period and Timeless; (2) Determine whether the rhythm in lark protein abundance results from diurnal changes in the translation of lark mRNA or through posttranslational alterations of protein stability; (3) Test the hypothesis that lark acts as a negative regulator of the clock output pathway mediating eclosion; (4) Pursue structure/function studies of the putative lark RNA-binding domains with the intent of defining which are relevant for the several functions of the gene; (5) Identify the in vivo mRNA targets of the lark protein. All of these experiments will address specific questions and test explicit hypotheses about the cellular and biochemical functions of lark protein. Although oscillating mRNAs and proteins have been documented in various organisms, lark represents the only functionally characterized circadian clock output component. In all organisms including humans, the clock regulation of rhythmic process must rely on comparable signaling pathways. Indeed, we have identified lark-gene homologues in mice and humans. Thus, Drosophila provides an excellent invertebrate model system for functional studies of circadian clock output pathways. In addition, the Drosophila system is also a good general model for the analysis of RNA-binding functions in an in vivo context. Finally, our proposed studies will yield reagents and experimental designs that should enhance the Drosophila system as a model for the study of other RNA-binding functions.